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Identifying mRNAs bound by RNA-binding proteins using affinity purification and differential display
Methods 26 (2002) 115–122 www.academicpress.com
Identifying mRNAs bound by RNA-binding proteins using affinity purification and differential display Nancy D. Rodgers, Xinfu Jiao, and Megerditch Kiledjian* Department of Cell Biology and Neuroscience, Nelson Biology Laboratories, Rutgers University, 604 Allison Road, Piscataway, NJ 08854, USA Accepted 10 January 2002
Abstract Many methods are available and widely used to determine specific proteins that bind to a particular RNA of interest. However, approaches to identify unknown substrate RNAs to which an RNA-binding protein binds and potentially regulates are not as common. In this article we describe a technique termed isolation of specific nucleic acids associated with proteins (SNAAP) that allows the identification of mRNAs associated with a protein. Methods are detailed for expressing and purifying fusion proteins that are used to isolate substrate mRNPs employing differential display technology. Lastly, experiments are described to confirm that the RNAs identified are indeed bonafide substrates for an RNA-binding protein. As the number of known RNA-binding proteins increases, of which many are involved in genetic disorders, it is essential that methodologies exist to identify RNA–protein interactions to better understand the manifestation of disease. Ó 2002 Elsevier Science (USA). All rights reserved.
1. Introduction The proper regulation of both mRNA stability and translation is essential for cell growth and survival. This regulation is controlled, in large part, by the binding of specific RNA-binding proteins to substrate mRNA. For example, the aCP proteins have been shown to be essential for the unusual stability of the a-globin mRNA [1]. Short-lived mRNAs, such as cytokine and growth factor transcripts, are thought to be bound by proteins that promote their instability [2]. RNA-binding proteins are also integral to translation. One of the best studied examples of translational regulation involves anterior and posterior definition in the Drosophila embryo where there are several examples of specific RNA-binding proteins that bind to and repress the translation of specific mRNAs [3,4]. Currently, a growing number of RNA-binding proteins are being identified and many of these proteins have been shown to be important in a number of different genetic disorders including azoospermia (lack of sperm production), fragile X mental retardation, and myotonic dystrophy [5–10]. However,
*
Corresponding author. Fax: +732-445-0104. E-mail address: [email protected] (M. Kiledjian).
the role of many of these proteins in specific diseases is unclear due in part to the lack of known RNA substrates for these proteins. The method most commonly used to identify a particular sequence to which an RNA-binding protein binds is SELEX (systematic evolution of ligands by exponential enrichment [11]). In this assay, multiple rounds of polymerase chain reaction (PCR) are used to amplify RNA bound by a particular RNA-binding protein. Eventually those RNAs that are bound with the highest affinity are selected from the starting pool of random RNAs. Although this is a powerful approach that has been used extensively, it has certain limitations. For example, the RNA sequence selected to contain the optimal binding site may not actually exist in vivo, leaving researchers with the task of identifying bonafide substrate mRNAs. We have developed a technique which we have termed isolation of specific nucleic acids associated with proteins (SNAAP) that identifies RNA substrates for any RNA-binding protein [12]. The technique combines a mRNA copurification assay with differential display. The pool of RNA substrates derives from cellular extract, that contains both RNA and protein. We have previously demonstrated that extract rather than purified RNA is required to ensure that the binding of a
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purified RNA-binding protein is specific [12]. Because all RNA-binding proteins possess some level of nonspecific binding, cellular proteins appear to provide a natural competition that selects RNAs bound with high affinity. Extract also consists of RNAs in their endogenous RNP context. The use of RNP allows for the identification of not only RNA substrates that are directly bound by the RNA-binding protein of interest (Fig. 1A) but also RNA substrates that are indirectly bound (Figs. 1B, C). An example in which collaborative binding to an mRNA is biologically critical is in the repression of hunchback (hb) mRNA translation by the nanos (Nos) protein in Drosophila. Nos is essential for hb repression; however, it is unable to recognize and bind to the hb mRNA in the absence of the pumilio protein [13].
The experimental procedures described here include the preparation of cellular extract from tissues and from cells, the expression and purification of RNA-binding proteins using both bacteria and insect cells, and the identification of RNA substrates for a specific protein. Methods that elucidate the binding site within an mRNA are also detailed. All methods are easily adapted to any protein and any source of cellular RNA. These techniques should greatly aid in furthering our understanding of the function of many RNA-binding proteins.
2. Description of methods 2.1. Preparation of extract The SNAAP technique involves the use of cellular extract that contains both the RNA substrates and any additional proteins that may be required for a particular RNA-binding protein to bind RNA. Any tissue may be used as a starting source provided that the particular protein of interest is expressed. Similarly whether to use total cellular extract or S130, which does not contain nuclei, depends on whether the protein being studied is nuclear or cytoplasmic. The following describes the isolation of total cellular extract from brain tissue and testis as well as the isolation of cytoplasmic S130 from tissue culture cells.
Fig. 1. Three possible-modes of RNA–protein interactions in which RNAs recovered from SNAAP may be involved. (A) Direct binding of an mRNA by an RNA-binding protein. The RNA is depicted by the solid line with a poly(A) tail and the RNA-binding protein of interest is shown as solid, black oval. (B) Collaborative Binding of the RNAbinding protein of interest is shown where a second, unknown protein is necessary for specific binding. In such a scenario, neither protein is competent to bind the particular RNA directly. The associated protein is depicted as a white square and may be made up of one or multiple proteins. (C) Interaction of the RNA-binding protein with another, unknown protein that is in direct contact with the RNA is the third possible interaction. The unknown protein is shown as a white circle. Although the RNA-binding protein of interest is not in direct contact with the RNA its association with the mRNP would indicate that it is important in some aspect of regulation of that mRNA.
2.1.1. Total extract from mouse brain A. We have routinely isolated brains from adult mice to prepare total cellular extract. The organ is washed twice in phosphate-buffered saline (PBS), sliced into small pieces with a razor, and disrupted with 20 strokes of a Potter-Elvehjem tissue homogenizer in lysis buffer. The resulting mixture is sonicated to lyse the cells and centrifuged at 15,000g for 15 min at 4° to pellet insoluble matter. The supernatant is collected, supplemented with glycerol to 5% (v/v), and frozen in aliquots at )70 °C. Any large tissue may be used to prepare extract but cells must be separated from each other with the use of a mechanical homogenizer. B. Smaller tissues such as testis and particular parts of a larger tissue may also be used. However, in this case it is only necessary to dice the tissue into small pieces with a razor blade and then proceed to the sonication step as described above. 2.1.2. S130 from cultured cells Cells are collected by centrifugation at 1000g for 3 min and washed twice with ice-cold PBS, after which the cell pellet can be stored at )70 °C. (The freezing step also aids in obtaining a more efficient lysis in the subsequent step.) The cells are resuspended in 1.5 ml of buffer A (see Buffers) per 108 cells and lysed with 25
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strokes of a type B pestle in a Dounce homogenizer. Nuclei are pelleted with a 4000g spin for 20 min and the resulting supernatant is layered over 2 ml of buffer A containing 30% (w/v) sucrose. The sample is centrifuged at 130,000g (31,000 K in a Beckman SW40 rotor) for 2 h, a thin layer of lipid that collects on the surface is aspirated off, and the supernatant is carefully removed without disturbing the S130–sucrose interface. The supernatant is adjusted to 5% glycerol (v/v) and frozen at )70 °C in aliquots. 2.2. Isolation of mRNA bound by the RNA-binding protein of interest The isolation of RNAs specifically bound by an RNAbinding protein requires that the protein and bound RNA can be separated from the pool of proteins and RNAs in the extract. To accomplish this, the protein must be immobilized. We describe two methods of expression and purification of recombinant protein from bacterial cells and from insect cells using a glutathione Stransferase (GST) tag and a myc-epitope tag, respectfully. The use of bacteria allows for the rapid preparation of a large quantity of recombinant protein; however, if posttranslational modifications are thought to be important for RNA binding, then the use of insect cells would be more appropriate. Theoretically any tag may be used to purify and immobilize recombinant protein; however, we have avoided the use of a hexahistidine (His6 ) tag since it is highly charged and may alter RNA binding. After recombinant protein has been purified, it is then incubated with the cellular extract of choice to allow interaction with and formation of mRNP complexes (Fig. 2). The protein, which is bound to an affinity matrix, is separated from the mixture and copurifying mRNA is then isolated and identified using differential display. Below we describe the generation and use of two different proteins implicated in human genetic disorders for which studies are ongoing in the laboratory. The first protein is murine deleted in azoospermia-like (mDAZL) protein which is a member of the DAZ family of proteins that are implicated in azoospermia [14–17]. The significance of mDAZL in spermatogenesis was demonstrated by a homozygous disruption of the gene in mice which resulted in the absence of germ cells in both sexes [18]. The DAZ family of proteins contains an RNP-motif RNA binding domain and binds RNA [19–21]. The second protein is the fragile X mental retardation protein 1 (FMRP). The absence of expression of FMRP is associated with fragile X syndrome, a common mental retardation syndrome [22]. FMRP contains two K homology (KH) domains and an RGG box and has been shown to bind RNA [23,24]. The function of neither of these proteins is understood in their respective disorders primarily because their RNA targets have yet to be defined.
Fig. 2. Schematic diagram of the copurification of mRNA with a fusion protein. On the left, the RNA-binding protein of interest (RBP) is tagged and immobilized on beads that bind specifically to the tag. The immobilized fusion protein is mixed with cell extract, depicted on the right, that consists of both additional proteins (shown as circles and squares) and mRNA substrates (shown as lines). After incubation, the beads are spun out of the solution and the resulting mRNP, is diagrammed on the bottom, is made up of the RBP and mRNA as well as an additional protein(s) although additional proteins do not necessarily have to be bound.
2.2.1. Expression of recombinant protein A. The mDAZL protein coding region is placed into the pGEX-6P-1 vector (Pharmacia, Piscataway, NJ) which allows bacterial expression and adds an N-terminal GST tag on the protein. The plasmid is transformed into Escherichia coli BL21 and grown to an OD600 of 0.5, and expression is induced with 0.2 mM isopropyl-b-D -thiogalactoside (IPTG) for 3 h at 30 °C. Cells expressing the fusion protein are resuspended in lysis buffer, disrupted by sonication, and spun at 31,000g for 10 min to pellet cellular debris. Bacterial RNAs are degraded by treating the extract with 200 U/ml micrococcal nuclease (Pharmacia) in the presence of 1 mM CaCl2 for 20 min at 30 °C. The reaction is stopped by the addition of 5 mM ethylene glycol bis(b-aminoethyl ether)-N ; N ; N 0 ; N 0 -tetraacetic acid (EGTA). B. The FMR1 protein coding region is PCR amplified with primers that introduced a myc-epitope tag at the 50 end and inserted into the pVL1393 vector (PharMingen). The plasmid is transfected into High Five insect cells (Invitrogen) which were grown at 27 °C in Grace’s
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Insect Medium (Gibco-BRL, Gaithersburg, MD) with 5% fetal bovine serum. Prior to transfection, the cells were seeded so that they would be approximately 50– 70% confluent on the day of transfection. Cells are counted and 2 106 cells are plated in one well of a sixwell plate and allowed to reattach for 10 min. The pVL1393-FMR1 plasmid (2 lg) is mixed with 0.1 lg of BaculoGold DNA (PharMingen) and incubated at room temperature for 5 min after which 1 ml of Transfection Buffer B (PharMingen) is added. The medium is aspirated from the cells and replaced with 1 ml of Transfection Buffer A (PharMingen) and the DNA/ Transfection Buffer B is added dropwise. The plate is incubated at 27 °C for 4 h after which the solution is aspirated and 3 ml of fresh medium is added. Viral particles are packed in the transfected cells and virus is released into the medium. However, the initial viral titer is low and must be amplified by subsequent infections to obtain large quantities of virus. Five days posttransfection, 0.5 ml of the medium is used lo infect a fresh, 60mm plate of cells that are 50–70% confluent. The plate is incubated at 27 °C for an additional 5 days after which 0.5 ml is used to infect a 100-mm plate of cells. After 5 days, >90% of the cells are infected and are floating and enlarged, two of the most obvious signs of infection. The medium is collected and kept as a stock solution at 4 °C to infect subsequent plates of cells that will be used for protein expression and purification. Plates of cells are infected with recombinant virus using 100 ll of the amplified viral stock. After four days the cells are collected, resuspended in lysis buffer (1 ml per 150-mm plate), sonicated, and spun at 15,000g for 30 min. The extract is then treated with micrococcal nuclease as described above for bacterially expressed proteins. 2.2.2. Copurification and isolation of mRNA bound to an RNA-binding protein The experiments detailed in the following sections all make use of the bacterially expressed, GST-tagged mDAZL; however, any tagged protein could easily be incorporated into the protocol. For example, antibody against the myc-tag on FMRP described above could have been immobilized with protein A–Sepharose and used in the following experiments as well. Bacterial extract containing approximately 50 lg of micrococcal nuclease-treated GST–mDAZL is immobilized on 40 ll of glutathione beads (Pharmacia) by incubating on a nutator for 15 min at 4 °C in RNA binding buffer with 0.5% Triton X-100 (RBB/0.5% TX). Any unbound protein is removed with four 1-ml washes in RBB/0.5% TX followed by two washes with 1 ml of RBB. Three hundred micrograms of testis total extract is precleared on glutathione beads (20 ll) in 350 ll of RBB for 10 min at 4 °C to remove RNAs that bind the beads nonspecifically. The beads are spun down and the resulting
extract is incubated with the fusion protein for 1 h at 4 °C, subsequently washed with 1 ml of RBB/0.25% TX, and then incubated in RBB/0.25% TX with 1 mg/ml heparin for 10 min at 4 °C. The heparin wash minimizes nonspecific RNA–protein interactions. The beads are washed for four times in RBB/0.25% TX and bound RNA is eluted from the beads by the addition of 200 ll of TE/1% sodium dodecyl sulfate (SDS) and boiled for 3 min. The RNA is phenol/chloroform (1/1) extracted, chloroform extracted twice, ethanol precipitated with 20 lg of glycogen (Roche) as a carrier, and washed with 70% ethanol. The RNA is resuspended in 15 ll of diethyl pyrocarbonate (DEPC)-treated H2 O and used for differential display. 2.2.3. Identification of mRNA by differential display The differential display is carried out under the recommendations of the manufacturer and using reagents supplied by GenHunter Corporation. The copurifying RNA is first subjected to reverse transcription (RT) with one of three different 30 primers that contain a stretch of 11 T nucleotides followed by an A, C, or G which can anneal to the poly(A) tail of each mRNA. In a total volume of 20 ll; 3 ll of RNA, 4 ll of 5 RT buffer, 1:6 ll of dNTPs ð250 lMÞ, and 2 ll of one of the 30 primers are denatured at 65 °C for 5 min and annealed at 37 °C for 10 min. MMLV reverse transcriptase (200 U, Promega, Madison, WI) is added and the mixture incubated for an additional 60 min at 37 °C to generate the cDNA, followed by a 5-min incubation at 75 °C to inactivate the enzyme. The RT products are used in the differential display PCRs with the same 30 primer used for the RT reaction and a subset of 80 different 50 primers. We often use a combination of four 50 primers as our pool of RNAs have already been greatly reduced by the copurification. This minimizes the number of PCRs as well as expedites the experiment by allowing the screening of a larger number of potential sequences in a shorter time. The reaction is set up in a total volume of 20 ll and contains 2 ll of RT product from above, 2 ll of 10 PCR buffer, 1:6 ll of dNTPs ð25 lMÞ, 2 ll of the same 30 primer used for the RT ð2 lMÞ, 2 ll each of the four 50 primers, 1 ll of ½a-35 SdATP (1250 Ci/ mmol), and 0:2 ll of AmpliTag (Perkin–Elmer Noswalk, CT). The PCR products are amplified with 30–40 cycles of denaturation at 94 °C for 30 seconds annealing at 40 °C for 2 min, and extension at 72 °C for 30 seconds, followed by a final extension for 5 min at 72 °C. The number of cycles needs to be determined empirically depending on the efficiency of the copurification and the abundance of the isolated mRNA. The radioactively labeled PCR products are separated on a 6% polyacrylamide/7 M urea denaturing gel and visualized by autoradiography. A representative example of the profile obtained using the GST-mDAZL protein is shown in Fig. 3. The first lane is the profile of total RNA
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bound by mDAZL but not the control proteins. From this experiment it appears that two RNAs that bind specifically to mDAZL have been identified and are represented by bands A and C. Neither of these bands is detected with aCP1 or RBD. Interestingly, neither band is detected in the PCR with total S130 extract (Fig. 3, lane 1), suggesting they are not abundant and were selectively enriched by mDAZL. The RNAs represented by bands B and D appear to be nonspecifically interacting with GST-mDAZL since they also copurified with the control RNA-binding proteins. After identification of bands representing RNAs specifically bound to the RNA-binding protein of interest, the band can be excised from the dry gel and the DNA eluted by soaking the gel slice in H2 O for 10 min and boiling for 15 min. The DNA is reamplified with the same 30 and 50 primers used in the differential display, cloned into the pGEM-T vector (Promega), and sequenced. 2.3. Confirmation of binding
Fig. 3. Profile of bands representing copurified RNAs from a differential display PCR. The RNA from a copurification with GSTmDAZL (lane 3), GST-aCP1 (lane 2), and GST-RBD (lane 4) was reverse transcribed with the H-T11 G 30 primer and used in a differential display PCR with the same 30 primer and a combination of H-AP 1–4 50 primers (GenHunter Corp.). Lane 1 shows the RT-PCR of total RNA isolated from testis total cell extract. Bands A and C represent mRNAs that are specifically bound by mDAZL, while bands B and D correspond to mRNAs that are nonspecifically associated with all the RNA-binding proteins tested.
used in the PCR. The profile of RNAs copurifying with GST-mDAZL is shown in lane 3 (Fig. 3). GST-aCP1 and the RNA binding domain of the hnRNP U protein (GST-RBD), which are both unrelated RNA-binding proteins, are also used in the SNAAP analysis to identify RNAs that nonspecifically bind to an RNA-binding protein (Fig. 3, lanes 2 and 4, respectively). A direct comparison of the bands that are present in the different lanes enables identification of bands that are specifically
After RNAs that appear to interact with an RNAbinding protein have been identified it is important to determine that the detected interaction is indeed real. We usually carry out SNAAP assays in duplicate and repeat the experiment at least twice, focusing on only those RNAs that are reproducibly copurified. The following section describes two commonly used methods for analyzing RNA–protein interactions: the electrophoretic mobility shift assay (EMSA) and a copurification assay. Other suitable methods such as a Northwestern blot can also be used. This requires that the protein is able to renature and bind RNA after being subjected to SDS–PAGE. It is important to keep in mind that SNAAP is able to detect both direct and indirect RNA–protein interactions. Also, the use of purified components may not always detect binding if additional proteins are necessary for efficient binding. The experiments described below use purified components consisting of mDAZL protein which we have previously shown can bind directly to a consensus binding site [21]. However, if binding is not detectable in these assays, it may be necessary to supplement the purified system with the extract used in the SNAAP procedure. It is possible that additional proteins may be required for binding to a particular RNA. 2.3.1. Electrophoretic mobility shift assay Using the SNAAP technique, we have previously demonstrated that the Trf2 mRNA is a substrate for mDAZL binding [21]. Mobility shift assays were carried out using 0.5 ng of in vitro-transcribed [32 P]UTP uniformly labeled Trf2 30 UTR RNA per reaction. The RNA was incubated with 2–6 lg of purified GSTmDAZL in RBB for 20 min at 25 °C in a total volume of 20 ll. Unbound RNA was degraded with RNase T1
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(1.5 units/20 ll, Roche) for 10 min at 25 °C. To disrupt nonspecific interactions, heparin competitor was added to the reaction to a final concentration of 2 mg/ml and incubated an additional 10 min. The RNase-resistant complexes were resolved on a 5% polyacrylamide gel (60:1 acrylamide:bisacrylamide) in 0.5 TBE buffer at 8 V/cm. A specific complex that forms between Trf2 RNA and mDAZL protein is shown in Fig. 4. (lanes 3–5). The complex becomes more intense with increasing amount of mDAZL protein and is specific since no binding is detected with an unrelated RNA binding protein (lanes 6–8). In instances where extract is used, many complexes may be detected due to additional proteins in the extract which could make it difficult to identify the specific complex containing the RNA-binding protein of interest. It may be possible to minimize many of these additional complexes by including RNase A (up to 10 ng) along with RNase T1 . Similarly, increased concentrations of heparin (up to 5 mg/ml) may help minimize background as well. Confirmation that the detected binding is specific can be assessed by competition assays. The reactions can be carried out as described above except in the presence of self or unrelated cold RNA. Single-stranded DNA could also be used instead of RNA if the protein can also bind DNA. Labeled RNA-to-competitor RNA ratios of 1:1, 1:10, and 1:100 are usually sufficient to see competition.
Fig. 4. Binding of GST-mDAZL to the Trf2 30 UTR. A 97-nucleotide segment (nucleotides 1177–1273) of the Trf2 30 UTR was used in an electrophoretic mobility shift assay using GST-mDAZL protein. The migration of the RNA probe is shown in lane 1 and RNA treated with RNase is shown in lane 2. RNA incubated with increasing amounts of GST-mDAZL from 2 to 6 lg is shown in lanes 3–5 and the resulting complex is labeled on the right. RNA was also incubated with an unrelated, nonspecific RNA-binding protein, RBD (lanes 6–8).
2.3.2. Copurification assay GST-mDAZL (4 lg) is bound to 20 ll of glutathione–sepharose beads for 15 min at 4 °C in RBB/0.5% TX. The beads are washed four times with 1 ml of RBB/ 0.5% TX and twice with 1 ml of RBB. In vitro-transcribed, 32 P-labeled RNA is incubated with the beads that contain the fusion protein in RBB/0.25% TX for 1 h at 4 °C, followed by four washes with RBB/0.25% TX. Bound RNA was eluted from the beads with 200 ll of TE/1% SDS and boiling for 2 min. The supernatant is phenol/choloroform extracted once and choloroform extracted twice followed by ethanol precipitation and a 70% ethanol wash. The RNA is resolved on a 5% polyacrylamide/7 M urea gel, dried, and exposed onto film, and the bound RNA visualized (please refer to Jiao et al. [21, Fig. 2] for a representative example). Control RNAs should be used in a parallel experiment to confirm that the detected binding is specific. If specificity needs to be improved it is possible to increase the stringency of the washes. This can be done by increasing the salt concentration in the RBB to up to 500 mM, by including 1 mg/ml heparin in the washes, and also by including up to 350 mM urea which often greatly reduces nonspecific interactions. 2.4. Identification of the binding site Once an RNA substrate has been confirmed for a particular RNA-binding protein it is important to determine the specific binding site within the RNA. This may allow the definition of a consensus RNA-binding sequence for that protein which could be used to determine if there are other RNA substrates. One straightforward strategy to begin narrowing down the binding site is to make three or more different RNAs that contain, e.g., the 50 untranslated region, the coding region, and the 30 untranslated region of the RNA. Radiolabeled RNAs corresponding to these regions can then be used in either EMSAs or copurification assays to determine where binding takes place. Then truncations of the binding region can be made from both the 50 and 30 ends to pinpoint the binding site. Alternatively, DNA oligonucleotides spanning portions of the RNA can be used in competition assays to determine which region of the RNA comprises the minimal binding site. Many RNA-binding proteins also bind DNA [12,21,25,26], which makes this type of experiment feasible. After a minimal binding site has been determined, nucleotides critical for binding can be identified by competition assays. Binding of protein to wild-type sequences can be carried out in the presence of competitor oligonucleotides that contain base substitutions. Mutations that compromise competition efficiency would correspond to sequences that are critical for binding (see Jiao et al. [21] for a representative example).
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2.5. Buffers 1 PBS: 137 mM NaCl, 2.7 mM KC1, 1.5 mM KH2 PO4 and 8.1 mM Na2 HPO4 7H2 O. Buffer A: 10 mM Tris, pH 7.5, 1 mM potassium acetate, 1.5 mM magnesium acetate, 2 mM dithiothreitol (DTT), 2 lg=ml leupeptin, and 0.5% aprotinin. Lysis buffer: 20 mM Hepes, pH 7.6, 1.5 mM MgCl2 , 10 mM KCl, 0.5 mM DTT, 2 lg=ml leupeptin, 0.5% aprotinin. RNA binding buffer (RBB): 10 mM Tris, pH 7.5, 1.5 mM MgCl2 , 250 mM KCl, 0.5 mM DTT, 2 lg=ml leupeptin, 0.5% aprotinin. 1 TBE: 89 mM Tris, 89 mM boric acid, 2 mM EDTA.
3. Concluding remarks The methods described in this report allow for the identification of RNA substrates for any given mRNAbinding protein. The techniques are quite pliable and can be altered to suit almost any need. For example, extract from virtually any tissue or cell type can be used in the experiment and since relatively little extract is required even small quantities of tissues or cells may be sufficient. Similarly, any RNA-binding protein can be used as long as it can be epitope-tagged, expressed, and purified from cells. The source of the recombinant protein could be bacteria or insect cells as outlined here or some other organism. The identification of unknown, bound RNAs described here uses differential display. Since the genome sequencing of many organisms is complete or nearly complete it should be possible to identify the gene of interest in most cases. However, often the gene may be novel and has to be cloned to confirm the interaction. With advances in mass spectroscopy, it may be possible in the near future to translate bound, copurifying mRNA and then identify the encoded proteins. The immunopurification strategy uses tagged recombinant protein with an antibody directed to the tag. An alternative approach is to use a direct monoclonal antibody specific for the endogenous protein of interest. Such an approach is described by Tenenbaum [27] in which identification of RNAs is accomplished by using microarrays. Both techniques have advantages and disadvantages. The use of recombinant protein relies on its ability to displace the endogenous protein in RNA– protein complexes facilitating the isolation of highaffinity sites. If endogenous protein is immunopurified using a monoclonal antibody, the problem of displacement is not an issue. A limitation of the direct antibody approach could be that the epitope is either not accessible in certain mRNP complexes and/or that the antibody reduces the stability of complexes. Purification of a
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protein with a tag would generally not encounter a similar problem. In both cases, it is essential that the tag or epitope recognized by the antibody be available to purify the protein. The identification of RNAs that bind proteins using microarrays is appealing because it is rapid and allows many transcripts to be screened at once. However, there are limits to the number of RNAs that can be represented on a particular glass slide, the costs may be high, and facilities for data analysis must be available. The use of differential display allows a costeffective method for screening (theoretically at least) a mammalian genome equivalent of mRNA to enable identification of novel genes that may be of interest. Both techniques will prove to be quite useful and should provide information on how particular RNA-binding proteins are involved in cellular metabolism. Acknowledgments This work was supported by NIH Grants HD39744 and DK51611 to M.K. References [1] Z. Wang, N. Day, P. Trifillis, M. Kiledjian, Mol. Cell. Biol. 19 (1999) 4552–4560. [2] C.Y. Chen, A.B. Shyu, Trends Biochem. Sci. 20 (1995) 465–470. [3] J. Dubnau, G. Struhl, Nature 379 (1996) 694–699. [4] D. Tautz, Nature 332 (1988) 281–284. [5] K. Ma, J.D. Inglis, A. Sharkey, W.A. Bickmore, R.E. Hill, E.J. Prosser, R.M. Speed, E.J. Thomson, M. Jobling, K. Taylor, J. Wolfe, H.J. Cooke, T.B. Hargreave, A.C. Chandley, Cell 75 (1993) 1287–1295. [6] M.C. Siomi, Y. Zhang, H. Siomi, G. Dreyfuss, Mol. Cell. Biol. 16 (1996) 3825–3832. [7] F.U. Mueller-Pillasch, U. Lacher, C. Wallrapp, A. Micha, F. Zimmerhackl, H. Hameister, G. Varga, H. Friess, M. Buchler, H.G. Beger, M.R. Vila, G. Adler, T.M. Gress, Oncogene 14 (1997) 2729–2733. [8] T.D. Levine, F. Gao, P.H. King, L.G. Andrews, J.D. Keene, Mol. Cell. Biol. 13 (1993) 3494–3504. [9] R.J. Buckanovich, Y.Y. Yang, R.B. Darnell, J. Neurosci. 16 (1996) 1114–1122. [10] L.T. Timchenko, N.A. Timchenko, C.T. Caskey, R. Roberts, Hum. Mol. Genet. 5 (1996) 115–121. [11] C. Tuerk, L. Gold, Science 249 (1990) 505–510. [12] P. Trifillis, N. Day, M. Kiledjian, RNA 5 (1999) 1071–1082. [13] J. Sonada, R.P. Wharton, Genes Dev. 13 (1999) 2704–2712. [14] E. Seboun, S. Barbaux, T. Bourgeron, S. Nishi, A. Agulnik, M. Egashira, N. Nikkawa, C. Bishop, M. Fellous, K. McElreavey, M. Kasahara, A. Algonik, Genomics 41 (1997) 227–235. [15] R. Saxena, L.G. Brown, T. Hawkins, R.K. Alagappan, H. Skaletsky, M.P. Reeve, R. Reijo, S. Rozen, M.B. Dinulos, C.M. Disteche, D.C. Page, Nat. Genet. 14 (1996) 292–299. [16] Z. Shan, P. Hirschmann, T. Seebacher, A. Edelmann, A. Jauch, J. Morell, P. Urbitsch, P.H. Vogt, Hum. Mol. Genet. 5 (1996) 2005–2011. [17] P.H. Yen, N.N. Chai, E.C. Salido, Hum. Mol. Genet. 5 (1996) 2013–2017. [18] M. Ruggiu, R. Speed, M. Taggart, S.J. McKay, F. Kilanowski, P. Saunders, J. Dorin, H.J. Cooke, Nature 389 (1997) 73–77.
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[23] C.T. Ashley Jr., K.D. Wilkinson, D. Reines, S.T. Warren, Science 262 (1993) 563–566. [24] H. Siomi, M.C. Siomi, R.L. Nussbaum, G. Dreyfuss, Cell 74 (1993) 291–298. [25] G. Dreyfuss, M.J. Matunis, S. Pinol-Roma, C.G. Burd, Annu. Rev. Biochem. 62 (1993) 289–321. [26] S. Pinol-Roma, Y.D. Choi, M.J. Matunis, G. Dreyfuss, Genes Dev. 2 (1998) 215–227. [27] S.A. Tenenbaum, P.J. Lager, C.C. Carson, J.D. Keene, Methods 26 (2002) 191–198.
Identifying mRNAs bound by RNA-binding proteins using affinity purification and differential display Nancy D. Rodgers, Xinfu Jiao, and Megerditch Kiledjian* Department of Cell Biology and Neuroscience, Nelson Biology Laboratories, Rutgers University, 604 Allison Road, Piscataway, NJ 08854, USA Accepted 10 January 2002
Abstract Many methods are available and widely used to determine specific proteins that bind to a particular RNA of interest. However, approaches to identify unknown substrate RNAs to which an RNA-binding protein binds and potentially regulates are not as common. In this article we describe a technique termed isolation of specific nucleic acids associated with proteins (SNAAP) that allows the identification of mRNAs associated with a protein. Methods are detailed for expressing and purifying fusion proteins that are used to isolate substrate mRNPs employing differential display technology. Lastly, experiments are described to confirm that the RNAs identified are indeed bonafide substrates for an RNA-binding protein. As the number of known RNA-binding proteins increases, of which many are involved in genetic disorders, it is essential that methodologies exist to identify RNA–protein interactions to better understand the manifestation of disease. Ó 2002 Elsevier Science (USA). All rights reserved.
1. Introduction The proper regulation of both mRNA stability and translation is essential for cell growth and survival. This regulation is controlled, in large part, by the binding of specific RNA-binding proteins to substrate mRNA. For example, the aCP proteins have been shown to be essential for the unusual stability of the a-globin mRNA [1]. Short-lived mRNAs, such as cytokine and growth factor transcripts, are thought to be bound by proteins that promote their instability [2]. RNA-binding proteins are also integral to translation. One of the best studied examples of translational regulation involves anterior and posterior definition in the Drosophila embryo where there are several examples of specific RNA-binding proteins that bind to and repress the translation of specific mRNAs [3,4]. Currently, a growing number of RNA-binding proteins are being identified and many of these proteins have been shown to be important in a number of different genetic disorders including azoospermia (lack of sperm production), fragile X mental retardation, and myotonic dystrophy [5–10]. However,
*
Corresponding author. Fax: +732-445-0104. E-mail address: [email protected] (M. Kiledjian).
the role of many of these proteins in specific diseases is unclear due in part to the lack of known RNA substrates for these proteins. The method most commonly used to identify a particular sequence to which an RNA-binding protein binds is SELEX (systematic evolution of ligands by exponential enrichment [11]). In this assay, multiple rounds of polymerase chain reaction (PCR) are used to amplify RNA bound by a particular RNA-binding protein. Eventually those RNAs that are bound with the highest affinity are selected from the starting pool of random RNAs. Although this is a powerful approach that has been used extensively, it has certain limitations. For example, the RNA sequence selected to contain the optimal binding site may not actually exist in vivo, leaving researchers with the task of identifying bonafide substrate mRNAs. We have developed a technique which we have termed isolation of specific nucleic acids associated with proteins (SNAAP) that identifies RNA substrates for any RNA-binding protein [12]. The technique combines a mRNA copurification assay with differential display. The pool of RNA substrates derives from cellular extract, that contains both RNA and protein. We have previously demonstrated that extract rather than purified RNA is required to ensure that the binding of a
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purified RNA-binding protein is specific [12]. Because all RNA-binding proteins possess some level of nonspecific binding, cellular proteins appear to provide a natural competition that selects RNAs bound with high affinity. Extract also consists of RNAs in their endogenous RNP context. The use of RNP allows for the identification of not only RNA substrates that are directly bound by the RNA-binding protein of interest (Fig. 1A) but also RNA substrates that are indirectly bound (Figs. 1B, C). An example in which collaborative binding to an mRNA is biologically critical is in the repression of hunchback (hb) mRNA translation by the nanos (Nos) protein in Drosophila. Nos is essential for hb repression; however, it is unable to recognize and bind to the hb mRNA in the absence of the pumilio protein [13].
The experimental procedures described here include the preparation of cellular extract from tissues and from cells, the expression and purification of RNA-binding proteins using both bacteria and insect cells, and the identification of RNA substrates for a specific protein. Methods that elucidate the binding site within an mRNA are also detailed. All methods are easily adapted to any protein and any source of cellular RNA. These techniques should greatly aid in furthering our understanding of the function of many RNA-binding proteins.
2. Description of methods 2.1. Preparation of extract The SNAAP technique involves the use of cellular extract that contains both the RNA substrates and any additional proteins that may be required for a particular RNA-binding protein to bind RNA. Any tissue may be used as a starting source provided that the particular protein of interest is expressed. Similarly whether to use total cellular extract or S130, which does not contain nuclei, depends on whether the protein being studied is nuclear or cytoplasmic. The following describes the isolation of total cellular extract from brain tissue and testis as well as the isolation of cytoplasmic S130 from tissue culture cells.
Fig. 1. Three possible-modes of RNA–protein interactions in which RNAs recovered from SNAAP may be involved. (A) Direct binding of an mRNA by an RNA-binding protein. The RNA is depicted by the solid line with a poly(A) tail and the RNA-binding protein of interest is shown as solid, black oval. (B) Collaborative Binding of the RNAbinding protein of interest is shown where a second, unknown protein is necessary for specific binding. In such a scenario, neither protein is competent to bind the particular RNA directly. The associated protein is depicted as a white square and may be made up of one or multiple proteins. (C) Interaction of the RNA-binding protein with another, unknown protein that is in direct contact with the RNA is the third possible interaction. The unknown protein is shown as a white circle. Although the RNA-binding protein of interest is not in direct contact with the RNA its association with the mRNP would indicate that it is important in some aspect of regulation of that mRNA.
2.1.1. Total extract from mouse brain A. We have routinely isolated brains from adult mice to prepare total cellular extract. The organ is washed twice in phosphate-buffered saline (PBS), sliced into small pieces with a razor, and disrupted with 20 strokes of a Potter-Elvehjem tissue homogenizer in lysis buffer. The resulting mixture is sonicated to lyse the cells and centrifuged at 15,000g for 15 min at 4° to pellet insoluble matter. The supernatant is collected, supplemented with glycerol to 5% (v/v), and frozen in aliquots at )70 °C. Any large tissue may be used to prepare extract but cells must be separated from each other with the use of a mechanical homogenizer. B. Smaller tissues such as testis and particular parts of a larger tissue may also be used. However, in this case it is only necessary to dice the tissue into small pieces with a razor blade and then proceed to the sonication step as described above. 2.1.2. S130 from cultured cells Cells are collected by centrifugation at 1000g for 3 min and washed twice with ice-cold PBS, after which the cell pellet can be stored at )70 °C. (The freezing step also aids in obtaining a more efficient lysis in the subsequent step.) The cells are resuspended in 1.5 ml of buffer A (see Buffers) per 108 cells and lysed with 25
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strokes of a type B pestle in a Dounce homogenizer. Nuclei are pelleted with a 4000g spin for 20 min and the resulting supernatant is layered over 2 ml of buffer A containing 30% (w/v) sucrose. The sample is centrifuged at 130,000g (31,000 K in a Beckman SW40 rotor) for 2 h, a thin layer of lipid that collects on the surface is aspirated off, and the supernatant is carefully removed without disturbing the S130–sucrose interface. The supernatant is adjusted to 5% glycerol (v/v) and frozen at )70 °C in aliquots. 2.2. Isolation of mRNA bound by the RNA-binding protein of interest The isolation of RNAs specifically bound by an RNAbinding protein requires that the protein and bound RNA can be separated from the pool of proteins and RNAs in the extract. To accomplish this, the protein must be immobilized. We describe two methods of expression and purification of recombinant protein from bacterial cells and from insect cells using a glutathione Stransferase (GST) tag and a myc-epitope tag, respectfully. The use of bacteria allows for the rapid preparation of a large quantity of recombinant protein; however, if posttranslational modifications are thought to be important for RNA binding, then the use of insect cells would be more appropriate. Theoretically any tag may be used to purify and immobilize recombinant protein; however, we have avoided the use of a hexahistidine (His6 ) tag since it is highly charged and may alter RNA binding. After recombinant protein has been purified, it is then incubated with the cellular extract of choice to allow interaction with and formation of mRNP complexes (Fig. 2). The protein, which is bound to an affinity matrix, is separated from the mixture and copurifying mRNA is then isolated and identified using differential display. Below we describe the generation and use of two different proteins implicated in human genetic disorders for which studies are ongoing in the laboratory. The first protein is murine deleted in azoospermia-like (mDAZL) protein which is a member of the DAZ family of proteins that are implicated in azoospermia [14–17]. The significance of mDAZL in spermatogenesis was demonstrated by a homozygous disruption of the gene in mice which resulted in the absence of germ cells in both sexes [18]. The DAZ family of proteins contains an RNP-motif RNA binding domain and binds RNA [19–21]. The second protein is the fragile X mental retardation protein 1 (FMRP). The absence of expression of FMRP is associated with fragile X syndrome, a common mental retardation syndrome [22]. FMRP contains two K homology (KH) domains and an RGG box and has been shown to bind RNA [23,24]. The function of neither of these proteins is understood in their respective disorders primarily because their RNA targets have yet to be defined.
Fig. 2. Schematic diagram of the copurification of mRNA with a fusion protein. On the left, the RNA-binding protein of interest (RBP) is tagged and immobilized on beads that bind specifically to the tag. The immobilized fusion protein is mixed with cell extract, depicted on the right, that consists of both additional proteins (shown as circles and squares) and mRNA substrates (shown as lines). After incubation, the beads are spun out of the solution and the resulting mRNP, is diagrammed on the bottom, is made up of the RBP and mRNA as well as an additional protein(s) although additional proteins do not necessarily have to be bound.
2.2.1. Expression of recombinant protein A. The mDAZL protein coding region is placed into the pGEX-6P-1 vector (Pharmacia, Piscataway, NJ) which allows bacterial expression and adds an N-terminal GST tag on the protein. The plasmid is transformed into Escherichia coli BL21 and grown to an OD600 of 0.5, and expression is induced with 0.2 mM isopropyl-b-D -thiogalactoside (IPTG) for 3 h at 30 °C. Cells expressing the fusion protein are resuspended in lysis buffer, disrupted by sonication, and spun at 31,000g for 10 min to pellet cellular debris. Bacterial RNAs are degraded by treating the extract with 200 U/ml micrococcal nuclease (Pharmacia) in the presence of 1 mM CaCl2 for 20 min at 30 °C. The reaction is stopped by the addition of 5 mM ethylene glycol bis(b-aminoethyl ether)-N ; N ; N 0 ; N 0 -tetraacetic acid (EGTA). B. The FMR1 protein coding region is PCR amplified with primers that introduced a myc-epitope tag at the 50 end and inserted into the pVL1393 vector (PharMingen). The plasmid is transfected into High Five insect cells (Invitrogen) which were grown at 27 °C in Grace’s
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Insect Medium (Gibco-BRL, Gaithersburg, MD) with 5% fetal bovine serum. Prior to transfection, the cells were seeded so that they would be approximately 50– 70% confluent on the day of transfection. Cells are counted and 2 106 cells are plated in one well of a sixwell plate and allowed to reattach for 10 min. The pVL1393-FMR1 plasmid (2 lg) is mixed with 0.1 lg of BaculoGold DNA (PharMingen) and incubated at room temperature for 5 min after which 1 ml of Transfection Buffer B (PharMingen) is added. The medium is aspirated from the cells and replaced with 1 ml of Transfection Buffer A (PharMingen) and the DNA/ Transfection Buffer B is added dropwise. The plate is incubated at 27 °C for 4 h after which the solution is aspirated and 3 ml of fresh medium is added. Viral particles are packed in the transfected cells and virus is released into the medium. However, the initial viral titer is low and must be amplified by subsequent infections to obtain large quantities of virus. Five days posttransfection, 0.5 ml of the medium is used lo infect a fresh, 60mm plate of cells that are 50–70% confluent. The plate is incubated at 27 °C for an additional 5 days after which 0.5 ml is used to infect a 100-mm plate of cells. After 5 days, >90% of the cells are infected and are floating and enlarged, two of the most obvious signs of infection. The medium is collected and kept as a stock solution at 4 °C to infect subsequent plates of cells that will be used for protein expression and purification. Plates of cells are infected with recombinant virus using 100 ll of the amplified viral stock. After four days the cells are collected, resuspended in lysis buffer (1 ml per 150-mm plate), sonicated, and spun at 15,000g for 30 min. The extract is then treated with micrococcal nuclease as described above for bacterially expressed proteins. 2.2.2. Copurification and isolation of mRNA bound to an RNA-binding protein The experiments detailed in the following sections all make use of the bacterially expressed, GST-tagged mDAZL; however, any tagged protein could easily be incorporated into the protocol. For example, antibody against the myc-tag on FMRP described above could have been immobilized with protein A–Sepharose and used in the following experiments as well. Bacterial extract containing approximately 50 lg of micrococcal nuclease-treated GST–mDAZL is immobilized on 40 ll of glutathione beads (Pharmacia) by incubating on a nutator for 15 min at 4 °C in RNA binding buffer with 0.5% Triton X-100 (RBB/0.5% TX). Any unbound protein is removed with four 1-ml washes in RBB/0.5% TX followed by two washes with 1 ml of RBB. Three hundred micrograms of testis total extract is precleared on glutathione beads (20 ll) in 350 ll of RBB for 10 min at 4 °C to remove RNAs that bind the beads nonspecifically. The beads are spun down and the resulting
extract is incubated with the fusion protein for 1 h at 4 °C, subsequently washed with 1 ml of RBB/0.25% TX, and then incubated in RBB/0.25% TX with 1 mg/ml heparin for 10 min at 4 °C. The heparin wash minimizes nonspecific RNA–protein interactions. The beads are washed for four times in RBB/0.25% TX and bound RNA is eluted from the beads by the addition of 200 ll of TE/1% sodium dodecyl sulfate (SDS) and boiled for 3 min. The RNA is phenol/chloroform (1/1) extracted, chloroform extracted twice, ethanol precipitated with 20 lg of glycogen (Roche) as a carrier, and washed with 70% ethanol. The RNA is resuspended in 15 ll of diethyl pyrocarbonate (DEPC)-treated H2 O and used for differential display. 2.2.3. Identification of mRNA by differential display The differential display is carried out under the recommendations of the manufacturer and using reagents supplied by GenHunter Corporation. The copurifying RNA is first subjected to reverse transcription (RT) with one of three different 30 primers that contain a stretch of 11 T nucleotides followed by an A, C, or G which can anneal to the poly(A) tail of each mRNA. In a total volume of 20 ll; 3 ll of RNA, 4 ll of 5 RT buffer, 1:6 ll of dNTPs ð250 lMÞ, and 2 ll of one of the 30 primers are denatured at 65 °C for 5 min and annealed at 37 °C for 10 min. MMLV reverse transcriptase (200 U, Promega, Madison, WI) is added and the mixture incubated for an additional 60 min at 37 °C to generate the cDNA, followed by a 5-min incubation at 75 °C to inactivate the enzyme. The RT products are used in the differential display PCRs with the same 30 primer used for the RT reaction and a subset of 80 different 50 primers. We often use a combination of four 50 primers as our pool of RNAs have already been greatly reduced by the copurification. This minimizes the number of PCRs as well as expedites the experiment by allowing the screening of a larger number of potential sequences in a shorter time. The reaction is set up in a total volume of 20 ll and contains 2 ll of RT product from above, 2 ll of 10 PCR buffer, 1:6 ll of dNTPs ð25 lMÞ, 2 ll of the same 30 primer used for the RT ð2 lMÞ, 2 ll each of the four 50 primers, 1 ll of ½a-35 SdATP (1250 Ci/ mmol), and 0:2 ll of AmpliTag (Perkin–Elmer Noswalk, CT). The PCR products are amplified with 30–40 cycles of denaturation at 94 °C for 30 seconds annealing at 40 °C for 2 min, and extension at 72 °C for 30 seconds, followed by a final extension for 5 min at 72 °C. The number of cycles needs to be determined empirically depending on the efficiency of the copurification and the abundance of the isolated mRNA. The radioactively labeled PCR products are separated on a 6% polyacrylamide/7 M urea denaturing gel and visualized by autoradiography. A representative example of the profile obtained using the GST-mDAZL protein is shown in Fig. 3. The first lane is the profile of total RNA
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bound by mDAZL but not the control proteins. From this experiment it appears that two RNAs that bind specifically to mDAZL have been identified and are represented by bands A and C. Neither of these bands is detected with aCP1 or RBD. Interestingly, neither band is detected in the PCR with total S130 extract (Fig. 3, lane 1), suggesting they are not abundant and were selectively enriched by mDAZL. The RNAs represented by bands B and D appear to be nonspecifically interacting with GST-mDAZL since they also copurified with the control RNA-binding proteins. After identification of bands representing RNAs specifically bound to the RNA-binding protein of interest, the band can be excised from the dry gel and the DNA eluted by soaking the gel slice in H2 O for 10 min and boiling for 15 min. The DNA is reamplified with the same 30 and 50 primers used in the differential display, cloned into the pGEM-T vector (Promega), and sequenced. 2.3. Confirmation of binding
Fig. 3. Profile of bands representing copurified RNAs from a differential display PCR. The RNA from a copurification with GSTmDAZL (lane 3), GST-aCP1 (lane 2), and GST-RBD (lane 4) was reverse transcribed with the H-T11 G 30 primer and used in a differential display PCR with the same 30 primer and a combination of H-AP 1–4 50 primers (GenHunter Corp.). Lane 1 shows the RT-PCR of total RNA isolated from testis total cell extract. Bands A and C represent mRNAs that are specifically bound by mDAZL, while bands B and D correspond to mRNAs that are nonspecifically associated with all the RNA-binding proteins tested.
used in the PCR. The profile of RNAs copurifying with GST-mDAZL is shown in lane 3 (Fig. 3). GST-aCP1 and the RNA binding domain of the hnRNP U protein (GST-RBD), which are both unrelated RNA-binding proteins, are also used in the SNAAP analysis to identify RNAs that nonspecifically bind to an RNA-binding protein (Fig. 3, lanes 2 and 4, respectively). A direct comparison of the bands that are present in the different lanes enables identification of bands that are specifically
After RNAs that appear to interact with an RNAbinding protein have been identified it is important to determine that the detected interaction is indeed real. We usually carry out SNAAP assays in duplicate and repeat the experiment at least twice, focusing on only those RNAs that are reproducibly copurified. The following section describes two commonly used methods for analyzing RNA–protein interactions: the electrophoretic mobility shift assay (EMSA) and a copurification assay. Other suitable methods such as a Northwestern blot can also be used. This requires that the protein is able to renature and bind RNA after being subjected to SDS–PAGE. It is important to keep in mind that SNAAP is able to detect both direct and indirect RNA–protein interactions. Also, the use of purified components may not always detect binding if additional proteins are necessary for efficient binding. The experiments described below use purified components consisting of mDAZL protein which we have previously shown can bind directly to a consensus binding site [21]. However, if binding is not detectable in these assays, it may be necessary to supplement the purified system with the extract used in the SNAAP procedure. It is possible that additional proteins may be required for binding to a particular RNA. 2.3.1. Electrophoretic mobility shift assay Using the SNAAP technique, we have previously demonstrated that the Trf2 mRNA is a substrate for mDAZL binding [21]. Mobility shift assays were carried out using 0.5 ng of in vitro-transcribed [32 P]UTP uniformly labeled Trf2 30 UTR RNA per reaction. The RNA was incubated with 2–6 lg of purified GSTmDAZL in RBB for 20 min at 25 °C in a total volume of 20 ll. Unbound RNA was degraded with RNase T1
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(1.5 units/20 ll, Roche) for 10 min at 25 °C. To disrupt nonspecific interactions, heparin competitor was added to the reaction to a final concentration of 2 mg/ml and incubated an additional 10 min. The RNase-resistant complexes were resolved on a 5% polyacrylamide gel (60:1 acrylamide:bisacrylamide) in 0.5 TBE buffer at 8 V/cm. A specific complex that forms between Trf2 RNA and mDAZL protein is shown in Fig. 4. (lanes 3–5). The complex becomes more intense with increasing amount of mDAZL protein and is specific since no binding is detected with an unrelated RNA binding protein (lanes 6–8). In instances where extract is used, many complexes may be detected due to additional proteins in the extract which could make it difficult to identify the specific complex containing the RNA-binding protein of interest. It may be possible to minimize many of these additional complexes by including RNase A (up to 10 ng) along with RNase T1 . Similarly, increased concentrations of heparin (up to 5 mg/ml) may help minimize background as well. Confirmation that the detected binding is specific can be assessed by competition assays. The reactions can be carried out as described above except in the presence of self or unrelated cold RNA. Single-stranded DNA could also be used instead of RNA if the protein can also bind DNA. Labeled RNA-to-competitor RNA ratios of 1:1, 1:10, and 1:100 are usually sufficient to see competition.
Fig. 4. Binding of GST-mDAZL to the Trf2 30 UTR. A 97-nucleotide segment (nucleotides 1177–1273) of the Trf2 30 UTR was used in an electrophoretic mobility shift assay using GST-mDAZL protein. The migration of the RNA probe is shown in lane 1 and RNA treated with RNase is shown in lane 2. RNA incubated with increasing amounts of GST-mDAZL from 2 to 6 lg is shown in lanes 3–5 and the resulting complex is labeled on the right. RNA was also incubated with an unrelated, nonspecific RNA-binding protein, RBD (lanes 6–8).
2.3.2. Copurification assay GST-mDAZL (4 lg) is bound to 20 ll of glutathione–sepharose beads for 15 min at 4 °C in RBB/0.5% TX. The beads are washed four times with 1 ml of RBB/ 0.5% TX and twice with 1 ml of RBB. In vitro-transcribed, 32 P-labeled RNA is incubated with the beads that contain the fusion protein in RBB/0.25% TX for 1 h at 4 °C, followed by four washes with RBB/0.25% TX. Bound RNA was eluted from the beads with 200 ll of TE/1% SDS and boiling for 2 min. The supernatant is phenol/choloroform extracted once and choloroform extracted twice followed by ethanol precipitation and a 70% ethanol wash. The RNA is resolved on a 5% polyacrylamide/7 M urea gel, dried, and exposed onto film, and the bound RNA visualized (please refer to Jiao et al. [21, Fig. 2] for a representative example). Control RNAs should be used in a parallel experiment to confirm that the detected binding is specific. If specificity needs to be improved it is possible to increase the stringency of the washes. This can be done by increasing the salt concentration in the RBB to up to 500 mM, by including 1 mg/ml heparin in the washes, and also by including up to 350 mM urea which often greatly reduces nonspecific interactions. 2.4. Identification of the binding site Once an RNA substrate has been confirmed for a particular RNA-binding protein it is important to determine the specific binding site within the RNA. This may allow the definition of a consensus RNA-binding sequence for that protein which could be used to determine if there are other RNA substrates. One straightforward strategy to begin narrowing down the binding site is to make three or more different RNAs that contain, e.g., the 50 untranslated region, the coding region, and the 30 untranslated region of the RNA. Radiolabeled RNAs corresponding to these regions can then be used in either EMSAs or copurification assays to determine where binding takes place. Then truncations of the binding region can be made from both the 50 and 30 ends to pinpoint the binding site. Alternatively, DNA oligonucleotides spanning portions of the RNA can be used in competition assays to determine which region of the RNA comprises the minimal binding site. Many RNA-binding proteins also bind DNA [12,21,25,26], which makes this type of experiment feasible. After a minimal binding site has been determined, nucleotides critical for binding can be identified by competition assays. Binding of protein to wild-type sequences can be carried out in the presence of competitor oligonucleotides that contain base substitutions. Mutations that compromise competition efficiency would correspond to sequences that are critical for binding (see Jiao et al. [21] for a representative example).
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2.5. Buffers 1 PBS: 137 mM NaCl, 2.7 mM KC1, 1.5 mM KH2 PO4 and 8.1 mM Na2 HPO4 7H2 O. Buffer A: 10 mM Tris, pH 7.5, 1 mM potassium acetate, 1.5 mM magnesium acetate, 2 mM dithiothreitol (DTT), 2 lg=ml leupeptin, and 0.5% aprotinin. Lysis buffer: 20 mM Hepes, pH 7.6, 1.5 mM MgCl2 , 10 mM KCl, 0.5 mM DTT, 2 lg=ml leupeptin, 0.5% aprotinin. RNA binding buffer (RBB): 10 mM Tris, pH 7.5, 1.5 mM MgCl2 , 250 mM KCl, 0.5 mM DTT, 2 lg=ml leupeptin, 0.5% aprotinin. 1 TBE: 89 mM Tris, 89 mM boric acid, 2 mM EDTA.
3. Concluding remarks The methods described in this report allow for the identification of RNA substrates for any given mRNAbinding protein. The techniques are quite pliable and can be altered to suit almost any need. For example, extract from virtually any tissue or cell type can be used in the experiment and since relatively little extract is required even small quantities of tissues or cells may be sufficient. Similarly, any RNA-binding protein can be used as long as it can be epitope-tagged, expressed, and purified from cells. The source of the recombinant protein could be bacteria or insect cells as outlined here or some other organism. The identification of unknown, bound RNAs described here uses differential display. Since the genome sequencing of many organisms is complete or nearly complete it should be possible to identify the gene of interest in most cases. However, often the gene may be novel and has to be cloned to confirm the interaction. With advances in mass spectroscopy, it may be possible in the near future to translate bound, copurifying mRNA and then identify the encoded proteins. The immunopurification strategy uses tagged recombinant protein with an antibody directed to the tag. An alternative approach is to use a direct monoclonal antibody specific for the endogenous protein of interest. Such an approach is described by Tenenbaum [27] in which identification of RNAs is accomplished by using microarrays. Both techniques have advantages and disadvantages. The use of recombinant protein relies on its ability to displace the endogenous protein in RNA– protein complexes facilitating the isolation of highaffinity sites. If endogenous protein is immunopurified using a monoclonal antibody, the problem of displacement is not an issue. A limitation of the direct antibody approach could be that the epitope is either not accessible in certain mRNP complexes and/or that the antibody reduces the stability of complexes. Purification of a
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protein with a tag would generally not encounter a similar problem. In both cases, it is essential that the tag or epitope recognized by the antibody be available to purify the protein. The identification of RNAs that bind proteins using microarrays is appealing because it is rapid and allows many transcripts to be screened at once. However, there are limits to the number of RNAs that can be represented on a particular glass slide, the costs may be high, and facilities for data analysis must be available. The use of differential display allows a costeffective method for screening (theoretically at least) a mammalian genome equivalent of mRNA to enable identification of novel genes that may be of interest. Both techniques will prove to be quite useful and should provide information on how particular RNA-binding proteins are involved in cellular metabolism. Acknowledgments This work was supported by NIH Grants HD39744 and DK51611 to M.K. References [1] Z. Wang, N. Day, P. Trifillis, M. Kiledjian, Mol. Cell. Biol. 19 (1999) 4552–4560. [2] C.Y. Chen, A.B. Shyu, Trends Biochem. Sci. 20 (1995) 465–470. [3] J. Dubnau, G. Struhl, Nature 379 (1996) 694–699. [4] D. Tautz, Nature 332 (1988) 281–284. [5] K. Ma, J.D. Inglis, A. Sharkey, W.A. Bickmore, R.E. Hill, E.J. Prosser, R.M. Speed, E.J. Thomson, M. Jobling, K. Taylor, J. Wolfe, H.J. Cooke, T.B. Hargreave, A.C. Chandley, Cell 75 (1993) 1287–1295. [6] M.C. Siomi, Y. Zhang, H. Siomi, G. Dreyfuss, Mol. Cell. Biol. 16 (1996) 3825–3832. [7] F.U. Mueller-Pillasch, U. Lacher, C. Wallrapp, A. Micha, F. Zimmerhackl, H. Hameister, G. Varga, H. Friess, M. Buchler, H.G. Beger, M.R. Vila, G. Adler, T.M. Gress, Oncogene 14 (1997) 2729–2733. [8] T.D. Levine, F. Gao, P.H. King, L.G. Andrews, J.D. Keene, Mol. Cell. Biol. 13 (1993) 3494–3504. [9] R.J. Buckanovich, Y.Y. Yang, R.B. Darnell, J. Neurosci. 16 (1996) 1114–1122. [10] L.T. Timchenko, N.A. Timchenko, C.T. Caskey, R. Roberts, Hum. Mol. Genet. 5 (1996) 115–121. [11] C. Tuerk, L. Gold, Science 249 (1990) 505–510. [12] P. Trifillis, N. Day, M. Kiledjian, RNA 5 (1999) 1071–1082. [13] J. Sonada, R.P. Wharton, Genes Dev. 13 (1999) 2704–2712. [14] E. Seboun, S. Barbaux, T. Bourgeron, S. Nishi, A. Agulnik, M. Egashira, N. Nikkawa, C. Bishop, M. Fellous, K. McElreavey, M. Kasahara, A. Algonik, Genomics 41 (1997) 227–235. [15] R. Saxena, L.G. Brown, T. Hawkins, R.K. Alagappan, H. Skaletsky, M.P. Reeve, R. Reijo, S. Rozen, M.B. Dinulos, C.M. Disteche, D.C. Page, Nat. Genet. 14 (1996) 292–299. [16] Z. Shan, P. Hirschmann, T. Seebacher, A. Edelmann, A. Jauch, J. Morell, P. Urbitsch, P.H. Vogt, Hum. Mol. Genet. 5 (1996) 2005–2011. [17] P.H. Yen, N.N. Chai, E.C. Salido, Hum. Mol. Genet. 5 (1996) 2013–2017. [18] M. Ruggiu, R. Speed, M. Taggart, S.J. McKay, F. Kilanowski, P. Saunders, J. Dorin, H.J. Cooke, Nature 389 (1997) 73–77.
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